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81.
Andreas Schmid Gerhard Burckhardt Heinz Gögelein 《The Journal of membrane biology》1989,111(3):265-275
Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl–=Br–=I–>SO
4
2–
F–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl–-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes. 相似文献
82.
Summary Cell-attached patch clamp recordings from unfertilized oocytes of the ascidianBoltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/m2, a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na+, Ca2+ and K+, but not Cl–. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca2+ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events. 相似文献
83.
R. H. Ashley 《The Journal of membrane biology》1989,111(2):179-189
Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k
d
3nm.B
max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl– and Ca2+, respectively. The reconstituted Ca2+ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca2+ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca2+/TRIS+ permeability ratio of 11.0. They also conducted other divalent cations (Ba2+>Ca2+>Sr2+>Mg2+) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca2+ channels, sharing some of the characteristics of Ca2+ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca2+ release from internal membrane stores. 相似文献
84.
Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K+ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH
i
. (1) The time course of K+(ATP) channel activity during exposure to and washout of NH4Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH4Cl entry and inhibition of the Na
o
+
H
i
+
exchanger, resembles the time course of pH
i
measured in other cell types that have been similarly treated. (2) Increasing pH
o
over the range 6.25–7.9 increases K+(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H+ by the application of the K+/H+ exchanger nigericin. (3) Increasing pH
i
over a similar range produces similar effects on K+(ATP) channels in inside-out excised patches exposed to small concentrations of ATP
i
. The physiological role of pH
i
in the metabolic gating of this channel remains to be explored. 相似文献
85.
Adam Bertl 《The Journal of membrane biology》1989,109(1):9-19
Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K+-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K+ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na+ and Cl–. However, addition of Na+ decreases the K+ currents. TheI–V relationships of the open channel as measured at various K+ concentrations with or without Na+ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data. 相似文献
86.
Summary We have investigated the ion permeability properties of sodium channels purified from eel electroplax and reconstituted into liposomes. Under the influence of a depolarizing diffusion potential, these channels appear capable of occasional spontaneous openings. Fluxes which result from these openings are sodium selective and blocked (from opposite sides of the membrane) by tetrodotoxin (TTX) and moderate concentrations of the lidocaine analogue QX-314. Low concentrations of QX-314 paradoxically enhance this channel-mediated flux. N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), reagents which remove inactivation gating in physiological preparations, transiently stimulate the sodium permeability of inside-out facing channels to high levels. The rise and subsequent fall of permeability appear to result from consecutive covalent modifications of the protein. Titration of the protein with the more reactive NBS can be used to produce stable, chronically active forms of the protein. Low concentrations of QX-314 produce a net facilitation of channel activation by NBA, while higher concentrations produce block of conductance. This suggests that rates of modifications by NBA which lead to the activation of permeability are influenced by conformational changes induced by QX-314 binding. 相似文献
87.
Summary Elementary Na+ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na+ channels.Iodate (10 mmol/liter) removed Na+ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na+ channels under control conditions, iodate-modified Na+ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na+ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na+ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na+ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na+ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na+ channels. 相似文献
88.
G. Tiger P.-E. Björklund R. F. Cowburn C. J. Fowler†‡ 《Journal of neurochemistry》1989,52(6):1843-1853
Increasing the [K+] in the assay medium from 5.7 to 17.8 mM produces a large enhancement of the inositol phospholipid breakdown response to the muscarinic agonist carbachol in rat cerebral cortical miniprisms, with minor effects on basal inositol phospholipid breakdown. This effect is also found with Rb+. The enhancement by a raised [K+] is not accompanied by a change in the composition of the labelled polyphosphoinositides. The carbachol-stimulated inositol phospholipid breakdown at 17.8 and 42.7 mM K+ was antagonised by veratrine (5-80 microM), 4-aminopyridine (5 mM), and tetraethylammonium (20 mM). These compounds, however, also inhibited the binding of [3H]quinuclidinyl benzilate to cortical membranes. BRL 34915 (0.2-20 microM) was without significant effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+.Mg2+ (10 mM) considerably reduced the carbachol-stimulated inositol phospholipid breakdown at 17.8, but not 42.7, mM K+. Inositol phospholipid breakdown was also stimulated, albeit to a small extent, by L-glutamate (100-3,000 microM) and quisqualate (1-100 microM), with the stimulation being additive to that produced by carbachol at both 5.7 and 17.8 mM K+. N-Methyl-D-aspartate (10-1,000 microM in Mg2+-free medium) had no significant effect on basal inositol phospholipid breakdown and had little or no effect on carbachol-stimulated inositol phospholipid breakdown at either 5.7 or 17.8 mM K+. It is concluded that it may not be correct to ascribe wholly the enhancement by K+ of carbachol-stimulated inositol phospholipid breakdown to the tissue-depolarising actions of this ion and that other actions of K+ may be involved. 相似文献
89.
Michael R. Blatt 《Planta》1990,180(3):445-455
Evidence of a role for abscisic acid (ABA) in signalling conditions of water stress and promoting stomatal closure is convincing, but past studies have left few clues as to its molecular mechanism(s) of action; arguments centred on changes in H+-pump activity and membrane potential, especially, remain ambiguous without the fundamental support of a rigorous electrophysiological analysis. The present study explores the response to ABA of K+ channels at the membrane of intact guard cells ofVicia faba L. Membrane potentials were recorded before and during exposures to ABA, and whole-cell currents were measured at intervals throughout to quantitate the steady-state and time-dependent characteristics of the K+ channels. On adding 10 M ABA in the presence of 0.1, 3 or 10 mM extracellular K+, the free-running membrane potential (V
m) shifted negative-going (–)4–7 mV in the first 5 min of exposure, with no consistent effect thereafter. Voltage-clamp measurements, however, revealed that the K+-channel current rose to between 1.84- and 3.41-fold of the controls in the steady-state with a mean halftime of 1.1 ± 0.1 min. Comparable changes in current return via the leak were also evident and accounted for the minimal response inV
m. Calculated atV
m, the K+ currents translated to an average 2.65-fold rise in K+ efflux with ABA. Abscisic acid was not observed to alter either K+-current activation or deactivation.These results are consistent with an ABA-evoked mobilization of K+ channels or channel conductance, rather than a direct effect of the phytohormone on K+-channel gating. The data discount notions that large swings in membrane voltage are a prerequisite to controlling guard-cell K+ flux. Instead, thev highlight a rise in membranecapacity for K+ flux, dependent on concerted modulations of K+-channel and leak currents, and sufficiently rapid to account generally for the onset of K+ loss from guard cells and stomatal closure in ABA. 相似文献
90.
Solubilization of Sodium Channel from Human Brain 总被引:1,自引:0,他引:1
Cécile de Rycker Christian Grandfils Lucien Bettendorff Ernest Schoffeniels 《Journal of neurochemistry》1989,52(2):349-353
[3H]Tetrodotoxin binds to a single class of receptor sites in homogenates of human brain with a KD of 9.1 nM at 0 degree C and a maximal binding capacity of 5.9 pmol/mg of protein. This tetrodotoxin receptor has been solubilized, and several parameters influencing the efficiency of this critical step have been studied. Treatment of brain membranes with 2% (wt/vol) Nonidet P-40 solubilizes up to 38% of the tetrodotoxin receptor sites. The duration of this solubilization step must not exceed 15 min at an optimal pH of 6.8. The binding activity is most stable when exogenous phosphatidylcholine is added to the soluble receptor with a phosphatidylcholine/detergent ratio of 1:5. 相似文献